HIV vaccine formulation

ABSTRACT

Immunogenic compositions containing a human immunodeficiency virus (HIV) gp140 protein, sorbitol, polysorbate 20, and histidine buffer are described. The described immunogenic compositions are advantageous in that they are stable at refrigerated temperature for extended periods of time, and are compatible with an adjuvant. Also described are methods of using the immunogenic compositions to induce an immune response against an HIV in a subject. The immunogenic compositions can be administered alone, or in combination with one or more additional HIV antigens, or one or more adenovirus vectors encoding the one or more additional HIV antigens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/623,684, filed on Jun. 15, 2017, which is entitled to prioritypursuant to 35 U.S.C. § 119(e) to U.S. Provisional Application No.62/350,919, filed on Jun. 16, 2016, the disclosures of which are hereinincorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “688097_115U2_Sequence Listing,” creation date of Mar. 5,2019, and having a size of 81 kb. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus (HIV) affects millions of people worldwide,and the prevention of HIV through an efficacious vaccine remains a veryhigh priority, even in an era of widespread antiretroviral treatment.HIV-1 is the most common and pathogenic strain of the virus, with morethan 90% of HIV/AIDS cases deriving from infection with HIV-1 group M.The M group is subdivided further into clades or subtypes. Anefficacious vaccine ideally would be capable of eliciting both potentcellular responses and broadly neutralizing antibodies capable ofneutralizing HIV-1 strains from different clades.

The high genetic variability of HIV-1 makes the development of a HIV-1vaccine an unprecedented challenge. In order to improve coverage ofpotential T-cell epitopes, and improve cellular responses, “mosaic”HIV-1 Gag, Pol and Env antigens, derived from HIV Group Antigen (Gag),Polymerase (Pol), and Envelope (Env) proteins, were described by othersand developed in an attempt to provide maximal coverage of potentialT-cell epitopes (e.g., Barouch et al, Nat Med 2010, 16: 319-323). Themosaic antigens are similar in length and domain structure to wild-type,naturally occurring HIV-1 antigens.

Sequences encoding mosaic antigens have been cloned in vectors, forexample, such as recombinant adenovirus serotype 26 (rAd26), and theserecombinant vectors have been used in vaccines to generate immuneresponses against HIV (see e.g. Barouch et al, supra; and WO2010/059732). Viral vectors expressing such mosaic HIV antigens haveproven to be effective in eliciting an immune response against HIVinfection.

Another therapeutic strategy that has been explored for inducing immuneresponses against HIV is the use of trimeric HIV envelope proteins asimmunogens in vaccines, such as gp140. The native envelope spike on thesurface of HIV is trimeric. Examples of trimeric envelope proteinsinclude clade C gp140 protein, and a mosaic envelope trimer protein,such as those disclosed in WO 2014/042942 and WO 2014/107744.

Clade C gp140 protein has previously been described e.g. in WO2010/042942 and in Nkolola et al. 2010, but there was no focus on anypharmaceutical formulation work in those disclosures. The protein was inphosphate-buffered saline (PBS) in some of the experiments in thosedisclosures. Mosaic gp140 has been described previously e.g. in WO2014/107744 and in Nkolola et al 2014, but again there was no focus onany pharmaceutical formulation work in those disclosures. The proteinwas in 25 mM Tris pH 7.5 and 150 mM NaCl in some of the experiments inthose disclosures.

Trimeric HIV envelope proteins, such as gp140, are capable of inducingpotent immune responses. Such envelope proteins can also be administeredin combination with other HIV antigens, such as mosaic antigens, toprovide enhanced immunity against HIV. However, the stability of the HIVenvelope proteins as trimers is not optimal under conditions typicallyused for clinical and commercial manufacturing. The trimeric HIVenvelope proteins are susceptible to both chemical and physicaldegradation. Moreover, many different factors, such as the bufferformulation, can affect the stability of proteins, and the effects areoften unpredictable. For example, the use of HEPES buffer in proteinformulations has been shown to result in generation of hydrogen peroxidewhen exposed to ambient light during the manufacturing process, whichcan impact the stability of the protein as well as other components inthe formulation, such as surfactants. See, e.g., Baicu et al.Cryobiology (2002) 45(1) 33-48; Lepe-Zuniga et al. J. Immunol. Methods(1987) 103(1), 145; and Zigler et al. In Vitro Cell. Dev. Biol. (1985)21(5), 282-287. It is desirable to have an HIV vaccine gp140 formulationthat would be suitable for stability of different variants of gp140protein, such as Clade C or mosaic gp140, and preferably with AluminumPhosphate adjuvant as a single vial drug product (rather than beingentirely dependent upon pharmacy mixing immediately prior to delivery ofthe vaccine), and in addition would enable drug product manufacturingmeeting large late phase and commercial scale demands. It is generallyunpredictable which combination of ingredients will result in aformulation that meets all these requirements.

Accordingly, there is a need in the art for improved formulations of HIVgp140 proteins with better stability under conditions used for clinicaland commercial manufacturing in order to realize the full therapeuticpotential of such trimeric envelope proteins. These formulations shouldalso be compatible for use with additional HIV antigen(s), includingvectors expressing HIV antigen(s), and/or adjuvants.

BRIEF SUMMARY OF THE INVENTION

The invention relates to immunogenic compositions of HIV gp140 proteinsthat have improved stability. The immunogenic compositions of theinvention can be stored under refrigerated conditions for extendedperiods of time, and are more optimal for use in clinical and commercialmanufacturing. The immunogenic compositions of the invention can alsoinclude an adjuvant. The invention also relates to methods of preparingthe immunogenic compositions, and methods of using the immunogeniccompositions to induce an immune response against HIV.

In one general aspect, the invention relates to an immunogeniccomposition comprising, relative to the total volume of the composition:

a. 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein;

b. 2% to 15% (w/v) sorbitol;

c. 0.01 to 0.05% (w/v) polysorbate 20; and

d. 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0.

Preferably, an immunogenic composition according to an embodiment of theinvention comprises a stabilized trimeric HIV gp140 protein, such as anHIV gp140 protein comprising the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2.

In particular embodiments of the invention, the immunogenic compositioncomprises 0.2 mg/mL to 1 mg/mL of an HIV gp140 protein.

In particular embodiments, an immunogenic composition according to anembodiment of the invention comprises 5% (w/v) to 12% (w/v) sorbitol.

In particular embodiments, the immunogenic composition comprises 0.02%(w/v) polysorbate 20.

In particular embodiments, the immunogenic composition comprises 10 mMhistidine buffer at a pH of 6.5.

According to embodiments of the invention, the immunogenic compositioncan further comprise an adjuvant, such as aluminum phosphate adjuvant.In certain of such embodiments, aluminum phosphate may be present in thecomposition at a concentration of 0.7-5.0 mg/mL, e.g. 0.8-4.0 mg/mL,e.g. 0.85 mg/mL, 1 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3.0 mg/mL,3.5 mg/mL, 3.84 mg/mL, 4.0 mg/mL. In certain embodiments, aluminumphosphate is present in the immunogenic compositions at a concentrationof 0.85 mg/mL. In certain embodiments, aluminum phosphate is present inthe immunogenic compositions at a concentration of 3.84 mg/mL.

In a preferred embodiment of the invention, an immunogenic compositioncomprises, relative to a total volume of the composition,

-   -   a. 0.2 mg/mL to 1 mg/mL of an HIV gp140 protein comprising the        amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2    -   b. 5% to 12% (w/v) sorbitol;    -   c. 0.02% (w/v) polysorbate 20;    -   d. 10 mM histidine buffer at a pH of 6.5; and    -   e. aluminum phosphate adjuvant, preferably at a concentration of        0.7-4.0 mg/mL.

In another general aspect, the invention relates to a method ofpreparing an immunogenic composition comprising admixing:

a. 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein;

b. 2% to 15% (w/v) sorbitol;

c. 0.01 to 0.05% polysorbate 20; and

d. 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0;

to thereby obtain the immunogenic composition.

In certain embodiments, the immunogenic composition of the inventioncomprises aluminum phosphate adjuvant, preferably at a concentration of0.7-4.0 mg/mL, and is stable upon storage at a temperature of 2-8° C.,for at least one month, preferably at least 2, 3, 4, 5, 6 months, morepreferably at least 7, 8, 9, 10, 11, 12 months, still more preferably atleast 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, mostpreferably at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36months or longer, e.g. 1-72 months, e.g. 6-48 months, e.g. 12-36 months.In certain embodiments, the immunogenic composition of the inventioncomprises aluminum phosphate adjuvant, preferably at a concentration of0.7-4.0 mg/mL, and is stable at a temperature of 25° C. for at least 6months, e.g. 6-12 months, or 6-24 months. In certain embodiments, theimmunogenic composition of the invention comprises aluminum phosphateadjuvant, preferably at a concentration of 0.7-4.0 mg/mL, and is stableat a temperature of 40° C. for at least 1 week, e.g. 1-12 weeks, e.g. atleast 2 weeks, 3 weeks, 4 weeks, 1 month, e.g. 1-2 months, 1-3 months,or 3-6 months.

And in another general aspect, the invention relates to a method ofinducing an immune response against a human immunodeficiency virus (HIV)in a subject in need thereof, comprising administering to the subject aneffective amount of an immunogenic composition of the invention.

In a particular embodiment, a method of inducing an immune responseagainst an HIV comprises administering to a subject in need thereof animmunogenic composition comprising, relative to the total volume of thecomposition,

-   -   a. 0.2 mg/mL to 1 mg/mL of an HIV gp140 protein comprising the        amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;    -   b. 5% to 12% (w/v) sorbitol;    -   c. 0.02% polysorbate 20;    -   d. 10 mM histidine buffer at a pH of 6.5; and    -   e. aluminum phosphate adjuvant, preferably at a concentration of        0.7-4.0 mg/mL.

In certain embodiments of the invention, a method of inducing an immuneresponse against an HIV further comprises administering to the subjectan effective amount of a second immunogenic composition comprising orencoding one or more additional HIV antigens, such as those comprisingor encoding an amino acid sequence of SEQ ID NOs: 3-12. Preferably, themethod comprises administering one or more vectors, preferablyadenovirus 26 vectors, encoding one or more HIV antigens, such as thosecomprising an amino acid sequence of SEQ ID NOs: 3-12. Methods ofinducing an immune response against an HIV can also compriseadministering one or more MVA vectors encoding one or more HIV antigens,such as those comprising an amino acid sequence of SEQ ID NOs: 3-12. Theone or more additional HIV antigens can also comprise SEQ ID NO: 11having one or more mutations selected from the group consisting of (i)I529P (Ile to Pro at position 529), (ii) K480E (Lys to Glu at position480), and (iii) a combination of EK479-480RRRR (i.e. replacing GluLys atposition 479 and 480 by four consecutive Arg residues), I529P (Ile toPro at position 529), A471C (Ala to Cys at position 471) and T575C (Thrto Cys at position 575). In one embodiment, the antigen comprising SEQID NO: 11 comprises SEQ ID NO: 12.

In another general aspect, the invention relates to a method forpreparing a long-term, storage stable immunogenic composition thatcomprises HIV gp140 protein, the method comprising:

(i) admixing the following components to create an immunogeniccomposition comprising these components in amounts relative to the totalvolume of the composition:

-   -   (a) 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein;    -   (b) 2% to 15% (w/v) sorbitol;    -   (c) 0.01 to 0.05% (w/v) polysorbate 20; and    -   (d) 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0,    -   (e) water,    -   (f) aluminum phosphate adjuvant, preferably at a concentration        of 0.7-4.0 mg/mL;        and        (ii) storing the composition at 2-8° C. for at least one month,        e.g. 1-72 months, e.g. 6-48 months, e.g. 12-36 months.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood that the invention is notlimited to the precise embodiments shown in the drawings.

In the figures:

FIGS. 1A and 1B show the results of SolvoVPE analysis of HIV gp140protein formulations prepared with acetate buffer, phosphate buffer, andhistidine buffer, and dynamic light scattering (DLS) analysis of HIVgp140 protein formulations prepared with sorbitol and sucrose; inparticular:

FIG. 1A shows the results of the SolvoVPE analysis; the change inabsorbance at 350 nm (turbidity) is plotted against the change inabsorbance at 280 nm (concentration); data points indicate the changebetween samples analyzed at time 0 (T₀) and after stressing the samplesat 40° C. for 24 hours (T₂₄); SolvoVPE analysis was performed asdescribed in Example 1;

FIG. 1B shows the results of the DLS analysis; the change in radius(R_(h)) from time 0 (T₀) at 20° C. to time 7 days (T_(7 days)) at 70° C.is plotted by sugar (sucrose vs. sorbitol); DLS analysis was performedas described in Example 1;

FIGS. 2A-2F show the results of high-performance size exclusionchromatography (HP-SEC) analysis of HIV gp140 formulations as describedin Example 2; the relative amount of hexamer, trimer and high/lowmolecular weight species of HIV clade C gp140 protein was determined byHP-SEC of samples analyzed at time 0 (T₀) and after stressing thesamples at 40° C. for 24 hours (T₂₄); “Tri+Hex” refers to the relativeamount of trimer and hexamer species of HIV clade C gp140 protein; “LMW”refers to low molecular weight species, e.g., cleavage or degradationproducts, in particular:

FIG. 2A shows the relative amount (%) of hexamer and trimer species ofHIV clade C gp140 protein in T₀ samples at different proteinconcentrations (0.2 mg/mL and 1.0 mg/mL);

FIG. 2B shows the relative amount (%) of hexamer and trimer species ofHIV gp140 clade C protein in T₀ samples at different concentrations ofsurfactant (0.02% and 0.1%); the data shown is a combination of datacollected from samples containing PS20 and samples containing PS80;

FIG. 2C shows the relative amount (%) of hexamer and trimer species ofHIV clade C gp140 protein in T₂₄ samples at different proteinconcentrations (0.2 mg/mL and 1.0 mg/mL);

FIG. 2D shows the relative amount (%) of hexamer and trimer species ofHIV gp140 clade C protein in T₂₄ samples at different concentrations ofsurfactant (0.02% and 0.1%); the data shown is a combination of datacollected from samples containing PS20 and samples containing PS80;

FIG. 2E shows the relative amount (%) of low molecular weight species ofHIV clade C gp140 protein based on surfactant type (PS20 versus PS80) inT₀ samples;

FIG. 2F shows the relative amount (%) of low molecular weight species ofHIV gp140 clade C protein in T₂₄ samples at different concentrations ofsurfactant;

FIGS. 3A and 3B show the results of stability studies of HIV gp140protein formulations according to embodiments of the invention; HIVmosaic gp140 protein formulations were subjected to multiple freeze-thawcycles as described in Example 3;

FIG. 3A shows the protein concentration after one, three, and fivefreeze-thaw cycles as determined by measuring the absorbance at 280 nm;and

FIG. 3B shows the turbity of the tested formulations after one, three,and five freeze-thaw cycles as determined by measuring the absorbance at350 nm; and

FIGS. 4A-4C show the results of stability studies of HIV clade C gp140protein formulations; HIV clade C gp140 protein formulations were storedat 25° C. and 60% relative humidity (RH), and 40° C. and 75% RH, bothwith and without aluminum phosphate adjuvant, and then tested using areduced SDS analytical method as described in Example 4;

FIG. 4A shows the relative protein concentration (%) of HIV clade Cgp140 formulations stored at 25° C. and 60% RH without any aluminumphosphate adjuvant;

FIG. 4B shows the relative protein concentration (%) of HIV clade Cgp140 formulations stored at 40° C. and 75% RH without any aluminumphosphate adjuvant;

FIG. 4C shows the relative protein concentration (%) of HIV clade Cgp140 formulations stored at 25° C. and 60% RH with aluminum phosphateadjuvant; and

FIG. 4D shows the relative protein concentration (%) of HIV clade Cgp140 formulations stored at 40° C. and 75% RH with aluminum phosphateadjuvant.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. For example, aconcentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, aconcentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).As used herein, the use of a numerical range expressly includes allpossible subranges, all individual numerical values within that range,including integers within such ranges and fractions of the values unlessthe context clearly indicates otherwise.

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human, to whom will be or has been administered animmunogenic composition according to embodiments of the invention. Theterm “mammal” as used herein, encompasses any mammal. Examples ofmammals include, but are not limited to, mice, rats, rabbits, guineapigs, monkeys, humans, etc., more preferably a human.

The invention generally relates to immunogenic compositions comprisingHIV gp140 protein and optionally adjuvant, methods of preparing andstoring such compositions, and methods of inducing an immune responseagainst HIV in a subject with the immunogenic compositions, alone or incombination with one or more additional HIV antigens, which arepreferably expressed by one or more vectors.

HIV Antigens

Human immunodeficiency virus (HIV) is a member of the genusLentivirinae, which is part of the family of Retroviridae. Two speciesof HIV infect humans: HIV-1 and HIV-2. HIV-1 is the most common strainof HIV virus, and is known to be more pathogenic than HIV-2. As usedherein, the terms “human immunodeficiency virus” and “HIV” refer to, butare not limited to, HIV-1 and HIV-2.

HIV is categorized into multiple clades with a high degree of geneticdivergence. As used herein, the term “HIV clade” or “HIV subtype” refersto related human immunodeficiency viruses classified according to theirdegree of genetic similarity. There are currently three groups of HIV-1isolates: M, N, and O. Group M (major strains) consists of at least tenclades, A through J. Group O (outer strains) can consist of a similarnumber of clades. Group N is a new HIV-1 isolate that has not beencategorized in either group M or O.

As used herein, the terms “HIV antigen,” “HIV antigenic protein,” “HIVantigenic polypeptide,” and “HIV immunogen” refer to a polypeptidecapable of inducing an immune response, e.g., a humoral and/or cellularmediated response, against HIV in a subject. The HIV antigen can be aprotein of HIV, a fragment or epitope thereof, or a combination ofmultiple HIV proteins or portions thereof, that can induce an immuneresponse or produce an immunity, e.g., protective immunity, against HIVin a subject.

Preferably, an antigen is capable of raising in a host a protectiveimmune response, e.g., inducing an immune response against a viraldisease or infection, and/or producing an immunity in (i.e.,vaccinating) a subject against a viral disease or infection, thatprotects the subject against the viral disease or infection. Forexample, the antigen can comprise a protein or fragments thereof fromHIV, such as the HIV gag, pol and env gene products.

An HIV antigen can be any HIV-1 or HIV-2 antigen or fragment thereof.Examples of HIV antigens include, but are not limited to gag, pol, andenv gene products, which encode structural proteins and essentialenzymes. Gag, pol, and env gene products are synthesized aspolyproteins, which are further processed into multiple other proteinproducts. The primary protein product of the gag gene is the viralstructural protein Gag polyprotein, which is further processed into MA,CA, SP1, NC, SP2, and P6 protein products. The pol gene encodes viralenzymes (Pol, polymerase), and the primary protein product is furtherprocessed into RT, RNase H, IN, and PR protein products. The env geneencodes structural proteins, specifically glycoproteins of the virionenvelope. The primary protein product of the env gene is gp160, which isfurther processed into gp120 and gp41.

In certain embodiments, the HIV antigen comprises an HIV Gag, Env, orPol antigen, or any antigenic portion or epitope or combination thereof,preferably an HIV-1 Gag, Env, or Pol antigen or any antigenic portion orepitope or combination thereof.

HIV antigens can also be mosaic HIV antigens. As used herein, “mosaicantigen” refers to a recombinant protein assembled from fragments ofnatural sequences. Mosaic antigens resemble natural antigens, but areoptimized to maximize the coverage of potential T-cell epitopes found inthe natural sequences, which improves the breadth and coverage of theimmune response. Mosaic HIV antigens for use with the invention arepreferably mosaic HIV-1 Gag, Pol, and/or Env antigens. Mosaic HIV Gag,Pol, and/or Env antigens are mosaic antigens comprising multipleepitopes derived from one or more of the Gag, Pol, and/or Envpolyprotein sequences of HIV. For example, a mosaic GagPol antigencomprises a mosaic Gag sequence and a mosaic Pol sequence.

Examples of mosaic HIV Gag, Pol, and/or Env antigens that can be used inthe invention include those described in, e.g., US20120076812; Barouchet al., Nat Med 2010, 16:319-323; and Barouch et al., Cell 155:1-9,2013, all of which are incorporated herein by reference in theirentirety. Preferably, mosaic HIV Gag, Pol, and/or Env antigens for usewith the invention include, but are not limited to, mos1Env (SEQ ID NO:3), mos2Env (SEQ ID NO: 4), mos1Pol (SEQ ID NO: 5), mos2Pol (SEQ ID NO:6), mos1Gag (SEQ ID NO: 7), mos2Gag (SEQ ID NO: 8), and combinationsthereof, for example mos1GagPol (SEQ ID NO: 9) and mos2GagPol (SEQ IDNO: 10). Other examples of mosaic HIV antigens include synthetic HIV Envproteins, which are non-naturally occurring HIV envelope proteinsoptimized to induce an immune response or provide an immunity againstone or more naturally occurring HIV strains, such as that comprising SEQID NO: 11, or SEQ ID NO: 11 having one or more mutations selected fromthe group consisting of (i) I529P (Ile to Pro at position 529), (ii)K480E (Lys to Glu at position 480), and (iii) a combination ofEK479-480RRRR (i.e. replacing GluLys at position 479 and 480 by fourconsecutive Arg residues), I529P (Ile to Pro at position 529), A471C(Ala to Cys at position 471) and T575C (Thr to Cys at position 575), orin preferred embodiments, the antigen comprising SEQ ID NO: 11 comprisesthe amino acid sequence of SEQ ID NO: 12, all as described inPCT/EP2016/081159 (filed on 15 Dec. 2016 in the name of Janssen Vaccines& Prevention B.V.), which is herein incorporated by reference in itsentirety.

HIV gp140 Protein

As used herein, the term “HIV gp140 protein” refers to an uncleavedectodomain of trimeric gp160 envelope protein, i.e., (gp160)₃.Embodiments of the invention relate to improved formulations of HIVgp140, preferably a trimer and/or hexamer of the gp140 subunits bound tothe ectodomain of the gp41 subunits lacking the gp41 transmembrane andcytoplasmic segments. The HIV env gene encodes the precursor proteingp160, which is proteolytically cleaved into the two mature envelopeglycoproteins gp120 and gp41. First, gp160 trimerizes to (gp160)₃ andthen undergoes cleavage into the two noncovalently associated proteinsgp120 and gp41 via a cleavage reaction mediated by a host cell protease,furin, at a sequence highly conserved in retroviral envelopeglycoprotein precursors. Viral entry is subsequently mediated by atrimer of gp120/gp41 heterodimers. Gp120 is the receptor bindingfragment, and binds to the CD4 receptor on a target cell that has such areceptor, such as, e.g., a T-helper cell. Gp41, which is non-covalentlybound to gp120, is the fusion fragment and provides the second step bywhich HIV enters the cell. Gp41 is originally buried within the viralenvelope, but when gp120 binds to a CD4 receptor, gp120 changes itsconformation causing gp41 to become exposed, where it can assist infusion with the host cell. HIV gp140 protein has been used as asurrogate for the native state of the cleaved, viral spike.

Expression of gp140 proteins has been described in several publications(e.g. Zhang et al., 2001; Sanders et al., 2002; Harris et al., 2011),and the protein can nowadays also be ordered from service providers, indifferent variants e.g. based on different HIV strains. A gp140 proteinaccording to the invention can have a cleavage site mutation so that thegp120 domain and gp41 ectodomain are covalently linked, or alternativelythe gp120 domain and gp41 ectodomain can be non-covalently linked (e.g.by a disulphur bridge as for instance in SOSIP variants). Gp140 proteinshave been used in various vaccination experiments (e.g. Nkolola et al.,2010, 2014; Kovacs et al., 2012; Barouch et al., 2015; Sanders et al.,2015).

According to embodiments of the invention, the HIV gp140 protein can bea homotrimer (e.g., trimers comprising three identical polypeptideunits), or a heterotrimer (e.g., trimers comprising three polypeptidesthat are not all identical). The HIV gp140 protein can also be ahexamer. Both trimer species and hexamer species of HIV gp140 proteinhave immunogenicity against HIV, or can induce immune responses againstHIV in vivo. Preferably, the HIV gp140 protein is a trimer, and morepreferably a homotrimer. An HIV gp140 protein can be a naturallyoccurring sequence, e.g., a sequence isolated from any HIV clade, suchas clade A, clade B, clade C, etc., or a mosaic gp140 protein. A “mosaicgp140 protein” contains multiple epitopes derived from one or more gp140sequences of one or more HIV clades.

Preferably, an HIV gp140 protein is a stabilized trimeric gp140 protein.A “stabilized trimeric gp140 protein” is a gp140 protein that can haveor can be modified to include a polypeptide sequence, such as atrimerization domain, that increases the stability of the trimericstructure, or it can be a gp140 protein that is modified to containmutations (as compared to natural gp140 sequences) that stabilize atrimeric structure, such as SOSIP and/or other mutations. Examples oftrimerization domains include, but are not limited to, the T4-fibritin“foldon” trimerization domain, e.g., that having the amino acid sequenceof GSGGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 13); the coiled-coiltrimerization domain derived from GCN4, e.g., that having the amino acidsequence of MKQIEDKIEEILSKIYHIENEIARIKKLIGEV (SEQ ID NO: 14); and thecatalytic subunit of E. coli aspartate transcarbamoylase as a trimertag. Such trimerization domains can be used to support stable trimerformation (see e.g. WO 2010/042942, Nkolola et al. 2010, WO 2014/107744,and Nkolola et al. 2014, for stabilized trimers of gp140 proteins). Astabilized trimeric gp140 protein for use in the invention can alsoinclude cleavage site mutations to enhance stability, e.g., in the furincleavage sites, and/or so-called SOSIP mutations (see, e.g. Sanders etal., 2002, 2015). A stabilized trimeric gp140 protein can be derivedfrom a gp140 protein isolated from any HIV clade, e.g., clade A, cladeB, clade C, etc. A stabilized trimeric gp140 protein can also be astabilized mosaic gp140 protein.

Exemplary HIV gp140 proteins that can be used in the invention includeHIV clade C gp140 protein (SEQ ID NO: 1), HIV mosaic gp140 protein (SEQID NO: 2), and combinations thereof. Both the HIV clade C gp140 protein(SEQ ID NO: 1) and HIV mosaic gp140 protein (SEQ ID NO: 2) arestabilized trimeric gp140 proteins comprising a T4-fibritin “foldon”trimerization domain.

Immunogenic Compositions

In a first aspect, the invention relates to an immunogenic compositioncomprising an HIV gp140 protein. An “immunogenic composition” as usedherein refers to a composition capable of inducing an immune response ina subject who has been or will be administered the composition. Animmunogenic composition can be a vaccine. A “vaccine” refers to acomposition that can provide protective immunity or a protective immuneresponse to a subject, or that can be used to vaccinate a subject.According to embodiments of the invention, any HIV gp140 protein knownin the art in view of the present disclosure can be used in animmunogenic composition of the invention. An immunogenic composition cancomprise one or more HIV gp140 proteins, such as one, two, or three HIVgp140 proteins.

Preferably, the immunogenic composition of the invention comprises astabilized trimeric gp140 protein. In one preferred embodiment, an HIVgp140 protein is an HIV clade C gp140 protein, such as that comprisingthe amino acid sequence of SEQ ID NO: 1. In another preferredembodiment, an HIV gp140 protein is a HIV mosaic gp140 protein, such asthat comprising the amino acid sequence of SEQ ID NO: 2.

In other embodiments, an immunogenic composition comprises both an HIVclade C gp140 protein and an HIV mosaic gp140 protein, such as thosecomprising SEQ ID NO: 1 and SEQ ID NO: 2. For example, an immunogeniccomposition can comprise a mixture of an HIV clade C gp140 proteincomprising the amino acid sequence of SEQ ID NO: 1 and an HIV mosaicgp140 protein comprising the amino acid sequence of SEQ ID NO: 2, e.g.,in a 1:1 ratio.

The immunogenic compositions of the invention also comprise water,preferably water for injection. It is added to the composition insufficient quantity (q.s.), depending on the volume of the compositionbeing prepared.

According to embodiments of the invention, an immunogenic compositioncomprises 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein, such as 0.05mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4mg/mL, or 5 mg/mL. In particular embodiments of the invention, animmunogenic composition comprises 0.2 mg/mL or 1 mg/mL of an HIV gp140protein, such as those comprising SEQ ID NO: 1 or SEQ ID NO: 2. In otherparticular embodiments of the invention, an immunogenic compositioncomprises a 0.05 mg/mL to 5 mg/mL of a mixture of HIV gp140 proteins,such as those comprising SEQ ID NO: 1 and SEQ ID NO: 2, e.g., in a 1:1ratio.

Immunogenic compositions of the invention further comprise sorbitol(sugar), polysorbate 20 (surfactant), and histidine buffer. Theinventors surprisingly discovered that the stability of the HIV gp140protein in a composition comprising histidine had improved stability ascompared to that in compositions comprising other amino acids. Theinventors also surprisingly discovered that inclusion of polysorbate 20in the composition further improved the stability of the HIV gp140protein in the composition. Typically, buffers used for proteinformulations contain a combination of acetate and sorbitol, or acombination of histidine and sucrose (see e.g., Uchiyama, BiochimicaBiophysica Acta (2014) 1844, 2041-2052). Histidine and sorbitol are notusually used in combination in a buffer for protein formulations. To thebest of the knowledge of the inventors, the combination of histidine andsorbitol with polysorbate 20 (surfactant) has not been used in anycommercial protein drug formulations. Therefore, it was surprising tosee that the combination of histidine and sorbitol improved thestability of the HIV gp140 protein.

According to embodiments of the invention, the concentration of sorbitolcan be in a range of 2% to 15% (w/v), such as 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% (w/v). In one preferredembodiment, the concentration of sorbitol is 5% (w/v). In anotherpreferred embodiment, the concentration of sorbitol is 12% (w/v).

According to embodiments of the invention, the concentration ofpolysorbate 20 can be in a range of 0.01% to 0.05% (w/v), such as 0.01%,0.02%, 0.03%, 0.04%, or 0.05% (w/v). In one preferred embodiment, theconcentration of polysorbate 20 is 0.02% (w/v).

According to embodiments of the invention, the concentration ofhistidine buffer is in a range of 5 mM to 20 mM, such as 5 mM, 10 mM, 15mM or 20 mM, and is preferably 10 mM. The pH of the histidine buffer isin a range of 5.5 to 7.0, such as 5.5, 6.0, 6.5, or 7.0, and ispreferably 6.5. In one preferred embodiment, the concentration of thehistidine buffer is 10 mM and the pH of the histidine buffer is 6.5. AnypH value described herein, is to be understood as being modified in allinstances by the term “about,” which, when used with reference to a pHvalue includes ±0.5 of the recited pH value. Unless specified otherwise,all pH values refer to the pH of the histidine buffer itself that isincluded in an immunogenic composition of the invention.

In certain embodiments of the invention, an immunogenic compositionfurther comprises an adjuvant. The terms “adjuvant” and “immunestimulant” are used interchangeably herein, and are defined as one ormore substances that cause stimulation of the immune system, or enhancean immune response. For example, an adjuvant can be used to enhance animmune response to an HIV gp140 protein and/or an immunogeniccomposition of the invention when administered alone or further incombination with one or more adenovirus vectors encoding one or more HIVantigens. Adjuvants suitable for use with the invention should be onesthat are potentially safe, well tolerated and effective in people, suchas, for instance QS-21, Iscomatrix, Detox-PC, MPL-SE, MoGM-CSF,TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I,GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, aluminum salts(e.g. aluminum hydroxide, and/or aluminum phosphate; an example of analuminum phosphate adjuvant is AdjuPhos, a sterilized aluminum phosphatewet gel suspension), Adjuplex, and MF59. Preferably, the adjuvant is analuminum phosphate adjuvant.

According to embodiments of the invention, an aluminum phosphateadjuvant can be included in an immunogenic composition at concentrationsof between about 0.7 and 5.0 mg/mL, for instance 0.7, 0.75, 0.8, 0.85,0.9, 0.95, or 1 mg/ml, e.g. at a fixed concentration of 0.85 mg/mL, orfor instance 1.5, 2.0, 2.5, 3.0, 3.5, 3.6, 3.7, 3.8, 3.85, 3.9, 3.95,4.0, 4.5, or 5.0 mg/mL, e.g. at a fixed concentration of 3.84 mg/mL. Onegoal of the invention was to provide long-term stable formulations withaluminum phosphate adjuvant present together with the gp140 proteinimmunogen. It was surprisingly found that the liquid formulations of theinvention that comprise HIV gp140, histidine buffer, sorbitol, andpolysorbate 20, are stable for at least 6 months at 2-8° C. Theseformulations were also found to be stable for at least 6 months atelevated temperatures of 25° C. In addition, these formulations wereeven found to be stable for at least 2 weeks, 1 month, or even up to 3months at 40° C., as measured by reduced SDS PAGE.

In an exemplary embodiment of the invention, an immunogenic compositioncomprises, relative to the total volume of the composition:

-   -   a. 0.2 mg/mL to 1 mg/mL of an HIV gp140 protein comprising the        amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;    -   b. 5% to 12% (w/v) sorbitol;    -   c. 0.02% (w/v) polysorbate 20;    -   d. 10 mM histidine buffer at a pH of 6.5; and    -   e. 0.7 to 4.0 mg/mL (e.g. 0.85 mg/mL or 3.84 mg/mL) aluminum        phosphate adjuvant.

Immunogenic compositions of the invention can be formulated in anymatter suitable for administration to a subject to facilitateadministration and improve efficacy, including, but not limited to, oral(enteral) administration and parenteral injections. The parenteralinjections for instance can include subcutaneous injection,intramuscular injection, or intradermal injection. Immunogeniccompositions of the invention can also be formulated for other routes ofadministration, e.g. transmucosal, rectal, sublingual administration,oral, or intranasal. Preferably, an immunogenic composition isformulated for intramuscular injection.

Immunogenic compositions of the invention are advantageous in that theHIV gp140 protein can be stably stored in liquid form at refrigeratedtemperature, for instance 2° C. to 8° C., for extended periods of time,such as about 2 years. In certain embodiments, the immunogeniccompositions include an adjuvant, for instance aluminum phosphateadjuvant. The immunogenic compositions containing adjuvant are alsocompatible with storage in liquid form under refrigerated conditions forextended periods of time. The immunogenic compositions of the inventionare also compatible with storage in lyophilized form. However, thecompositions of the invention are thus preferably liquid formulations,meaning that they are in liquid form at the preferred storagetemperature, i.e. at 2-8° C. Typically the liquid formulations orcompositions according to the invention are aqueous suspensions, meaningthat not all protein and/or particulate material such as aluminumphosphate may be entirely dissolved. In such cases it is advised to mixthe composition before use. It is preferred to store the compositions ofthe invention that comprise aluminum phosphate adjuvant above thefreezing point of water, most preferably at 2-8° C., e.g. e.g. 2° C., 3°C., 4° C., 5° C., 6° C., 7° C., or 8° C. Advantages are that noresource-intensive and costly lyophilization and associatedre-dissolving before use is needed, no bed-side mixing and separatestorage of the aluminum phosphate adjuvant is needed but rather theformulations can be stored ‘ready-for-use’. In addition, storage atrefrigerated but not frozen conditions makes that the compositions canbe used more easily in resource-limited settings e.g. where no freezingcapacity is available. Moreover, the observed maintained stability atelevated temperatures (e.g. 25° C., and even 40° C.) of the compositionsof the invention indicates that inadvertent temperature excursions, e.g.when temporarily the composition is exposed to room temperature even inwarm climates, should not immediately be detrimental to the vaccinecomposition of the invention.

The compositions of the invention, surprisingly including the onescomprising aluminum phosphate, are stable upon storage at a temperatureof 2-8° C. for at least one day, one week, two weeks, one month,preferably at least 2, 3, 4, 5, 6 months. More preferably thesecompositions are stable under these conditions for at least 7, 8, 9, 10,11, 12 months, still more preferably at least 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 months, most preferably at least 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36 months or longer, e.g. 1-72 months, e.g.6-48 months, e.g. 12-36 months. The invention in certain embodimentsthus provides compositions according to the invention, which are stablewhen stored at 2-8° C. for at least one month, at least three months, atleast 6 months. Preferably such compositions are stable for at least 12months, at least 18 months, at least 24 months, at least 30 months, atleast 36 months. In certain embodiments, the invention provides usingthe immunogenic compositions of the invention for vaccinating a subject,preferably a human subject, after the immunogenic compositions have beenstored at 2-8° C. for at least one day, at least one week, at least twoweeks, at least three weeks, at least one month, at least two months, atleast three months, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. 28. 29, 30, 31, 32, 33,34, 35, 36 months.

For the purpose of the present invention, an immunogenic compositionthat comprises HIV gp140 protein and that after having been stored forat least one week, preferably at least one month, at a given temperaturedoes not show more than 25%, preferably not more than 10%, ofdegradation of said gp140 protein on a reduced SDS PAGE gel, isconsidered a long-term storage stable immunogenic composition at saidtemperature. An immunogenic composition that comprises HIV gp140 proteinis considered “stable” according to the present invention under certainconditions (e.g. 2-8° C.) for a specified time (e.g. 6 months), if underthese conditions after said specified time said gp140 protein does notshow more than 25%, preferably less than 20%, preferably less than 15%,preferably less than 10%, most preferably less than 5%, of degradation(compared to initial measurement at t=0) on a reduced SDS PAGE gel.Degradation is visible as additional bands below the gp140 band ofdesired molecular weight. Alternative assays such as ELISA can also beused to measure stability, and in certain embodiments, a compositionthat is stable as defined above, also does not show more than 50%,preferably less than 25%, degradation (reduction as compared to initialsignal at t=0) in an ELISA assay.

Methods of Preparing an Immunogenic Composition

The invention also relates to a method of preparing an immunogeniccomposition of the invention. According to embodiments of the invention,a method of preparing an immunogenic composition comprises admixing anHIV gp140 protein, sorbitol, polysorbate 20, and histidine buffer in theappropriate concentration ranges. One of ordinary skill in the art willbe familiar with conventional techniques used to prepare suchcompositions.

For example, immunogenic compositions can be prepared by mixinghistidine buffer, sorbitol, and polysorbate 20 at the desiredconcentrations. Then, the HIV gp140 protein can be added. The pH of thecomposition can be adjusted before or after addition of the HIV gp140protein. As another illustrative example, first a buffer solutioncontaining histidine and sorbitol can be prepared. The HIV gp140 proteinis prepared in buffer at the desired concentration, and then added tothe buffer solution containing histidine and sorbitol, followed byaddition of Polysorbate at the target concentration. Adjuvant can beadded last to obtain the final composition. The invention also providesmethods for preparing a long-term, storage stable immunogeniccomposition that comprises HIV gp140 protein. In certain embodiments,such methods comprise: (i) providing an immunogenic compositionaccording to the invention (i.e. comprising 0.05-5 mg/mL HIV gp140protein, 2-15% (w/v) sorbitol, 0.01-0.05% polysorbate 20, 5-20 mMhistine buffer pH 5.5-7.0, water, and preferably 0.7-4.0 mg/mL aluminumphosphate), and (ii) storing said composition at 2-8° C. for at leastone month, e.g. 1-72 months, e.g. 6-48 months, e.g. 12-36 months, e.g.18-30 months. In certain embodiments, such methods comprise:

(i) admixing the following components to create an immunogeniccomposition comprising these components in amounts relative to the totalvolume of the composition:

-   -   (a) 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein;    -   (b) 2% to 15% (w/v) sorbitol;    -   (c) 0.01 to 0.05% (w/v) polysorbate 20;    -   (d) 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0,    -   (e) water (preferably water for injection),    -   (f) aluminum phosphate adjuvant, preferably at a concentration        of 0.7-4 mg/mL; and        (ii) storing the composition at 2-8° C. for at least one month,        e.g. 1-72 months, e.g. 6-48 months, e.g. 12-36 months, e.g.        18-30 months.

Methods of Inducing an Immune Response

The invention also relates to methods of inducing an immune responseagainst human immunodeficiency virus (HIV) in a subject in need thereofwith an immunogenic composition of the invention. The immune responsecan be against one or more HIV clades. The methods described herein alsoinclude administering an immunogenic composition of the invention incombination with one or more additional HIV antigens that are preferablyexpressed from one or more vectors, such as adenovirus vectors or MVAvectors, including methods of priming and boosting an immune response.

In one general aspect, a method of inducing an immune response against ahuman immunodeficiency virus (HIV) in a subject in need thereofcomprises administering to the subject an effective amount of animmunogenic composition comprising an HIV gp140 protein according to anembodiment of the invention. Any of the immunogenic compositionsdescribed herein can be used in a method of inducing an immune responseagainst HIV in a subject. Preferably, the composition comprises astabilized trimeric HIV gp140 protein, such as an HIV clade C gp140protein comprising the amino acid sequence of SEQ ID NO: 1, or an HIVmosaic gp140 protein comprising the amino acid sequence of SEQ ID NO: 2,or a mixture thereof. The composition can further comprise an adjuvant,such as aluminum phosphate adjuvant. An exemplary embodiment of animmunogenic composition for use in the methods of the inventioncomprises, relative to the total volume of the composition,

-   -   a. 0.2 mg/mL to 1 mg/mL of an HIV gp140 protein comprising the        amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;    -   b. 5% to 12% (w/v) sorbitol;    -   c. 0.02% (w/v) polysorbate 20;    -   d. 10 mM histidine buffer at a pH of 6.5; and    -   e. 0.7 to 4.0 mg/mL (e.g. 0.85 mg/mL or 3.84 mg/mL) aluminum        phosphate adjuvant.

According to embodiments of the invention, “inducing an immune response”when used with reference to the methods and compositions describedherein encompasses providing protective immunity and/or vaccinating asubject against an infection, such as an HIV infection, for prophylacticpurposes, as well as causing a desired immune response or effect in asubject in need thereof against an infection, such as an HIV infection,for therapeutic purposes. Preferably, the methods of the invention arefor prophylactic purposes, such as for providing protective immunity.The immune response can be a cellular immune response and/or a humoralimmune response.

As used herein, the term “protective immunity” or “protective immuneresponse” means that the vaccinated subject is able to control aninfection with the pathogenic agent against which the vaccination wasdone. Usually, the subject having developed a “protective immuneresponse” develops only mild to moderate clinical symptoms or nosymptoms at all. Usually, a subject having a “protective immuneresponse” or “protective immunity” against a certain agent will not dieas a result of the infection with said agent.

Typically, administration of immunogenic compositions according toembodiments of the invention will have a prophylactic aim to generate animmune response against an HIV antigen before infection or developmentof symptoms. In other embodiments, the immunogenic compositions can beadministered for post-exposure prophylactics. Immunogenic compositionsof the invention can also be administered to a non-human mammal, such asfor experimental purposes.

As used herein, “an effective amount” or “immunologically effectiveamount” means an amount of a composition sufficient to induce a desiredimmune effect or immune response in a subject in need thereof. In oneembodiment, an effective amount means an amount sufficient to induce animmune response in a subject in need thereof. In certain embodiments, aneffective amount means an amount sufficient to produce immunity in asubject in need thereof, e.g., provide a protective effect against adisease such as a viral infection. In certain embodiments, an effectiveamount means an amount sufficient to enhance an immune response in asubject in need thereof. For example, when used in combination with oneor more other components or immunogenic compositions capable ofeffecting an immune response, such as in a prime-boost regimen, aneffective amount can be an amount sufficient to enhance the immuneresponse induced by the one or more other components or immunogeniccompositions.

An effective amount can vary depending upon a variety of factors, suchas the physical condition of the subject, age, weight, health, etc.; theparticular application, e.g., whether inducing immune response orproviding protective immunity; and the particular disease, e.g., viralinfection, for which immunity is desired. An effective amount canreadily be determined by one of ordinary skill in the art in view of thepresent disclosure. As general guidance, when used with reference to anHIV gp140 protein an effective amount can range from, e.g. about 0.3 toabout 3000 microgram (μg), e.g. 1-1000 μg, e.g. 10-500 μg, e.g. about50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μg.

An effective amount of an immunogenic composition can be administered ina single composition, or in multiple compositions, such as 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules and/orinjectables), wherein the administration of the multiple compositions(e.g., tablets, capsules and/or injectables) collectively provides asubject with the immunogenically effective amount. It is also possibleto administer an effective amount of an immunogenic composition to asubject, and subsequently administer another dose of an effective amountof an immungenic composition to the same subject, in a so-calledprime-boost regimen, as described in greater detail below.

According to embodiments of the invention, an immunogenic compositioncan be administered to a subject by any means known in the artincluding, but not limited to, oral (enteral) administration andparenteral injections. The parenteral injections could for instanceinclude subcutaneous injection, intramuscular injection, or intradermalinjection. Preferably, the immunogenic composition is administered byintramuscular injection.

It is also possible to administer immunogenic compositions of theinvention together with one or more additional HIV antigens, or one ormore vectors, such as adenovirus vectors, encoding one or moreadditional HIV antigens. As used herein, the terms “co-delivery,”“co-administration,” “administered together with,” or “administered incombination with” refer to simultaneous administration of two or morecomponents, such as an immunogenic composition comprising an HIV gp140protein, or multiple viral expression vectors, such as adenovirusvectors. “Simultaneous administration” can be administration of the twoor more components at least within the same day. When two components are“administered together with,” they can be administered in separatecompositions sequentially within a short time period, such as 24, 20,16, 12, 8, or 4 hours, or within 1 hour or less, or they can beadministered in a single composition at the same time. Non-limitingexamples of administration of immunogenic compositions of gp140 proteinwith one or more additional HIV antigens encoded by vectors such asadenoviral vectors, are provided in WO 2016/049287, the disclosure ofwhich is herein incorporated by reference in its entirety.

Thus, in certain embodiments of the invention, a method of inducing animmune response further comprises administering to the subject aneffective amount of a second immunogenic composition comprising one ormore HIV antigens or one or more vectors encoding the HIV antigens. AnyHIV antigen known to those skilled in the art in view of the presentdisclosure can be used, such as HIV Nef, Gag, Env, or Pol antigens orany antigenic portion or epitope or combination thereof. Mosaic HIVantigens can also be used. Exemplary HIV antigens include, but are notlimited to, mosaic Env, Gag, and/or Pol antigens and combinationsthereof, such as those comprising the amino acid sequences of SEQ IDNOs: 3-12, or SEQ ID NO: 11 having one or more mutations selected fromthe group consisting of (i) I529P (Ile to Pro at position 529), (ii)K480E (Lys to Glu at position 480), and (iii) a combination ofEK479-480RRRR (i.e. replacing GluLys at position 479 and 480 by fourconsecutive Arg residues), I529P (Ile to Pro at position 529), A471C(Ala to Cys at position 471) and T575C (Thr to Cys at position 575). Theadditional HIV antigens can for instance be administered to the subjectas isolated proteins or polypeptides or as vectors encoding theseproteins or polypeptides.

Preferably, in the method comprising administering to the subject aneffective amount of a second immunogenic composition, the secondimmunogenic composition comprises one or more vectors encoding one ormore HIV antigens.

Any vector known to those skilled in the art in view of the presentdisclosure can be used. Preferably, the vector is an adenovirus vector,more preferably an adenovirus 26 vector. The preparation of recombinantadenoviral vectors is well known in the art. Preparation of recombinantadenovirus 26 (rAd26) vectors is described, for example, in WO2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplarygenome sequences of Ad26 are found in GenBank Accession EF 153474 and inSEQ ID NO:1 of WO 2007/104792. Examples of vectors useful for theinvention for instance include those described in WO2012/082918, thedisclosure of which is incorporated herein by reference in its entirety.Other adenovirus vectors that can be used in combination withimmunogenic compositions of the invention include those described in WO2016/049287 and PCT Application No. PCT/EP2016/081159, the disclosuresof which are herein incorporated by reference in their entirety.

According to embodiments of the invention, adenovirus vectors cancomprise one HIV antigen, or more than one HIV antigen, such as two,three, or four or more HIV antigens. Immunogenic compositions cancomprise one or more adenovirus vectors, such as two, three, four ormore HIV antigens, encoding one or more different HIV antigens. Alsoaccording to embodiments of the invention, a second composition cancomprise one adenovirus vector, or more than one adenovirus vector, suchas two, three, four or more adenovirus vectors. If a second compositioncomprises more than one adenovirus vector, the adenovirus vectors canencode the same or different HIV antigens.

Adenovirus vectors encoding one or more HIV antigens for use in themethods of the invention comprise nucleic acid encoding an HIV antigenthat is operably linked to a promoter, meaning that the nucleic acid isunder the control of a promoter. The promoter can be a homologouspromoter (i.e., derived from the same genetic source as the vector) or aheterologous promoter (i.e., derived from a different vector or geneticsource). Examples of suitable promoters include the cytomegalovirus(CMV) promoter and the Rous Sarcoma virus (RSV) promoter. Preferably,the promoter is located upstream of the nucleic acid within anexpression cassette.

As general guidance, an “effective amount” when used with reference toadenovirus vectors, can range from about 10⁸ viral particles to about10¹² viral particles, for example 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² viralparticles. The preparation and use of immunogenic compositionscomprising adenovirus vectors, such as adenovirus 26 vectors, are wellknown to those of ordinary skill in the art. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolcan also be included.

For instance recombinant adenovirus vector can be stored in the bufferthat is also used for the Adenovirus World Standard (Hoganson et al.,2002, Bioprocessing J 1: 43-8): 20 mM Tris pH 8, 25 mM NaCl, and 2.5%glycerol. Another useful adenovirus formulation buffer suitable foradministration to humans is 20 mM Tris, 2 mM MgCl₂, 25 mM NaCl, 10%(w/v) sucrose, and 0.2% (w/v) polysorbate-80. Another formulation bufferthat is suitable for recombinant adenovirus comprises 10-25 mM citratebuffer pH 5.9-6.2, 4-6% (w/w) hydroxypropyl-beta-cyclodextrin (HBCD),70-100 mM NaCl, 0.018-0.035% (w/w) polysorbate-80, and optionally0.3-0.45% (w/w) ethanol. Other buffers can be used, and several examplesof suitable formulations for the storage and for pharmaceuticaladministration of purified vectors are known.

Administration of immunogenic compositions comprising the one or moreadditional HIV antigens or one or more adenovirus vectors encoding theone or more additional HIV antigens is typically intramuscular,intradermal or subcutaneous. However, other modes of administration suchas intravenous, rectal, cutaneous, oral, nasal, etc. can be envisaged aswell. Intramuscular administration of the immunogenic compositions canbe achieved by using a needle to inject a suspension of the expressionvectors, e.g. adenovirus vectors, and/or antigenic polypeptides. Analternative is the use of a needleless injection device to administerthe composition (using, e.g., Biojector™) or a freeze-dried powdercontaining the vaccine.

In one embodiment, an immunogenic composition comprising a gp140 proteinaccording to an embodiment of the invention is used in combination withan adenovirus vector, preferably an adenovirus 26 vector, encoding a HIVantigen comprising the amino acid sequence of SEQ ID NO: 3.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 4.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 5.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 6.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 7.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 8.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 9.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 10.

In one embodiment, an immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention is used incombination with an adenovirus vector, preferably an adenovirus 26vector, encoding a HIV antigen comprising the amino acid sequence of SEQID NO: 11, SEQ ID NO: 11 having one or more mutations selected from thegroup consisting of (i) I529P (Ile to Pro at position 529), (ii) K480E(Lys to Glu at position 480), and (iii) a combination of EK479-480RRRR(i.e. replacing GluLys at position 479 and 480 by four consecutive Argresidues), I529P (Ile to Pro at position 529), A471C (Ala to Cys atposition 471) and T575C (Thr to Cys at position 575), or SEQ ID NO: 12.

Upon administration of adenovirus vectors encoding one or more HIVantigens, the adenovirus vector expresses the encoded HIV antigens, suchthat the HIV antigens are presented to the immune system of the subject,thereby inducing the required response to produce immunity, or induce animmune response. An immunogenic composition comprising an HIV gp140protein according to an embodiment of the invention can be administeredtogether with the one or more adenovirus vectors to prime the immuneresponse, and/or subsequent to administration of the one or moreadenovirus vectors to boost the immune response.

Thus, in other embodiments of the invention, a method of inducing animmune response against an HIV in a subject in need thereof comprises(i) administering to the subject an effective amount of an immunogeniccomposition comprising an HIV gp140 protein according to an embodimentof the invention, and preferably further comprising an adjuvant, and(ii) administering to the subject an effective amount of a secondimmunogenic composition comprising one or more adenovirus vectorsencoding one or more HIV antigens. Steps (i) and (ii) are conducted ineither order, with one of the steps for priming immunization and theother for boosting immunization. Optionally, the method can furthercomprise administering one or more Modified Vaccinia Ankara (MVA)vectors encoding one or more HIV antigens. MVA vectors can encode anyHIV antigen described herein. Preferably, the MVA vectors encode one ormore HIV antigens selected from the group consisting of SEQ ID NOs:3-12, or SEQ ID NO: 11 having one or more mutations selected from thegroup consisting of (i) I529P (Ile to Pro at position 529), (ii) K480E(Lys to Glu at position 480), and (iii) a combination of EK479-480RRRR(i.e. replacing GluLys at position 479 and 480 by four consecutive Argresidues), I529P (Ile to Pro at position 529), A471C (Ala to Cys atposition 471) and T575C (Thr to Cys at position 575).

Examples of adenovirus vectors, MVA vectors, and prime-boost regimensthat can be used in combination with an immunogenic compositioncomprising an HIV gp140 protein according to an embodiment of theinvention include those described in WO 2016/049287, the disclosure ofwhich is herein incorporated by reference in its entirety.

For example, in one embodiment of the disclosed methods, one or moreadenovirus vectors encoding one or more HIV antigens are used to primethe immune response. An immunogenic composition comprising an HIV gp140protein according to the invention can be used together with the one ormore adenovirus vectors for the priming immunization. The primingimmunization can be administered only once, but can optionally also beadministered multiple times, for example, initial priming administrationat time 0, followed by another priming administration about 4-14 weeks,e.g. 10-14 weeks after the initial priming administration. Theimmunogenic composition comprising an HIV gp140 protein optionallytogether with one or more additional adenovirus or MVA vectors encodingone or more additional HIV antigens can be used to boost the immuneresponse. A boosting immunization can also be administered once ormultiple times, for example, first at about 22-26 weeks after theinitial priming administration, followed by another boostingadministration at about 46-50 weeks after the initial primingadministration. The immune response induced by the immunization ismonitored.

In other general aspects, the invention relates to vaccine combinationsfor inducing an immune response against a human immunodeficiency virus(HIV) in a subject in need thereof. According to embodiments of theinvention, the vaccine combination comprises an immunogenic compositioncomprising an HIV gp140 protein, such as that comprising the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2; one or more adenovirusvectors, preferably adenovirus 26 vectors, encoding one or more HIVantigens, such as an HIV antigen comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:s 3 to 12, and SEQ IDNO: 11 having one or more mutations selected from the group consistingof (i) I529P (Ile to Pro at position 529), (ii) K480E (Lys to Glu atposition 480), and (iii) a combination of EK479-480RRRR (i.e. replacingGluLys at position 479 and 480 by four consecutive Arg residues), I529P(Ile to Pro at position 529), A471C (Ala to Cys at position 471) andT575C (Thr to Cys at position 575) and optionally one or more MVAvectors encoding one or more HIV antigens, such as an HIV antigencomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:s 3 to 12, and SEQ ID NO: 11 having one or more mutationsselected from the group consisting of (i) I529P (Ile to Pro at position529), (ii) K480E (Lys to Glu at position 480), and (iii) a combinationof EK479-480RRRR (i.e. replacing GluLys at position 479 and 480 by fourconsecutive Arg residues), I529P (Ile to Pro at position 529), A471C(Ala to Cys at position 471) and T575C (Thr to Cys at position 575).

According to embodiments of the invention, the vaccine combinations canbe used in any of the methods described herein for inducing an immuneresponse against an HIV in a subject in need thereof, including forpriming and boosting an immune response.

EMBODIMENTS

Embodiment 1 is an immunogenic composition comprising, relative to thetotal volume of the composition:

a. 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein;

b. 2% to 15% (w/v) sorbitol;

c. 0.01 to 0.05% (w/v) polysorbate 20; and

d. 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0.

Embodiment 2 is the immunogenic composition of embodiment 1, wherein theconcentration of the HIV gp140 is 0.2 mg/mL.

Embodiment 3 is the immunogenic composition of embodiment 1, wherein theconcentration of the HIV gp140 is 1 mg/mL.

Embodiment 4 is the immunogenic composition of any of embodiments 1 to3, wherein the concentration of sorbitol is 5% (w/v).

Embodiment 5 is the immunogenic composition of any of embodiments 1 to3, wherein the concentration of sorbitol is 12% (w/v).

Embodiment 6 is the immunogenic composition of any of embodiments 1-5,wherein the concentration of polysorbate 20 is 0.02% (w/v).

Embodiment 7 is the immunogenic composition of any of embodiments 1-6,wherein the concentration of the histidine buffer is 10 mM, and the pHof the histidine buffer is 6.5.

Embodiment 8 is the immunogenic composition of any of embodiments 1-7,further comprising aluminum phosphate adjuvant, for instance at aconcentration of 0.7-1.0 mg/mL, preferably at a concentration of 0.85mg/mL.

Embodiment 9 is the immunogenic composition of any of embodiments 1-8,wherein the HIV gp140 protein comprises the amino acid sequence of SEQID NO: 1.

Embodiment 10 is the immunogenic composition of any of embodiments 1-8,wherein the HIV gp140 protein comprises the amino acid sequence of SEQID NO: 2.

Embodiment 11 is an immunogenic composition comprising, relative to thetotal volume of the composition,

-   -   a. 0.2 mg/mL to 1 mg/mL of an HIV gp140 protein comprising the        amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;    -   b. 5% to 12% (w/v) sorbitol;    -   c. 0.02% (w/v) polysorbate 20;    -   d. 10 mM histidine buffer at a pH of 6.5; and    -   e. aluminum phosphate adjuvant, e.g. at a concentration of        0.7-4.0 mg/mL, e.g. 0.85 mg/mL or 3.84 mg/mL.

Embodiment 12 is the immunogenic composition of embodiment 11,comprising 0.2 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 1 and 5% (w/v) sorbitol.

Embodiment 13 is the immunogenic composition of embodiment 11,comprising 0.2 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 2 and 5% (w/v) sorbitol.

Embodiment 14 is the immunogenic composition of embodiment 11,comprising 0.2 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 1 and 12% (w/v) sorbitol.

7 Embodiment 15 is the immunogenic composition of embodiment 11,comprising 0.2 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 2 and 12% (w/v) sorbitol.

Embodiment 16 is the immunogenic composition of embodiment 11,comprising 1.0 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 1 and 5% (w/v) sorbitol.

Embodiment 17 is the immunogenic composition of embodiment 11,comprising 1.0 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 2 and 5% (w/v) sorbitol.

Embodiment 18 is the immunogenic composition of embodiment 11,comprising 1.0 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 1 and 12% (w/v) sorbitol.

Embodiment 19 is the immunogenic composition of embodiment 11,comprising 1.0 mg/mL HIV gp140 protein comprising the amino acidsequence of SEQ ID NO: 2 and 12% (w/v) sorbitol.

Embodiment 20 is an immunogenic composition according to any ofembodiments 1 to 19 formulated for intramuscular injection.

Embodiment 21 is a method of preparing an immunogenic compositioncomprising admixing:

a. 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein;

b. 2% to 15% (w/v) sorbitol;

c. 0.01 to 0.05% polysorbate 20; and

d. 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0;

to thereby obtain the immunogenic composition.

Embodiment 22 is a method of inducing an immune response against an HIVin a subject in need thereof, comprising administering to the subject aneffective amount of an immunogenic composition comprising, relative tothe total volume of the composition,

a. 0.05 mg/mL to 5 mg/mL of an HIV gp140 protein;

b. 2% to 15% (w/v) sorbitol;

c. 0.01 to 0.05% polysorbate 20; and

d. 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0.

Embodiment 23 is the method of embodiment 22, wherein the immunogeniccomposition comprises, relative to the total volume of the composition,

-   -   a. 0.2 mg/mL to 1 mg/mL of an HIV gp140 protein comprising the        amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;    -   b. 5% to 12% (w/v) sorbitol;    -   c. 0.02% polysorbate 20;    -   d. 10 mM histidine buffer at a pH of 6.5; and    -   e. aluminum phosphate adjuvant, e.g. at a concentration of        0.7-4.0 mg/mL, e.g. 0.85 mg/mL or 3.84 mg/mL.

Embodiment 24 is a method of inducing an immune response against an HIVin a subject in need thereof, comprising administering to the subject aneffective amount of an immunogenic composition according to any one ofembodiments 1 to 20, or to any one of embodiments 36-37.

Embodiment 25 is the method of any one of embodiments 22 to 24, furthercomprising administering to the subject an effective amount of one ormore adenovirus vectors, preferably adenovirus 26 vectors, encoding oneor more HIV antigens.

Embodiment 26 is the method of any one of embodiments 22 to 25 furthercomprising administering to the subject an effective amount of one ormore MVA vectors encoding one or more HIV antigens.

Embodiment 27 is the method of embodiment 25 or embodiment 26, whereinthe one or more HIV antigens comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 3 to 12, and SEQ ID NO: 11having one or more mutations selected from the group consisting of (i)I529P (Ile to Pro at position 529), (ii) K480E (Lys to Glu at position480), and (iii) a combination of EK479-480RRRR (i.e. replacing GluLys atposition 479 and 480 by four consecutive Arg residues), I529P (Ile toPro at position 529), A471C (Ala to Cys at position 471) and T575C (Thrto Cys at position 575).

Embodiment 28 is an immunogenic composition according to any one ofembodiments 1-20, or to any one of embodiments 36-37, which is stable at2-8° C. for at least six months.

Embodiment 29 is an immunogenic composition according to any one ofembodiments 1-20, or to any one of embodiments 36-37, which is stable at2-8° C. for at least 12 months, at least 18 months, at least 24 months,at least 30 months, at least 36 months.

Embodiment 30 is an immunogenic composition according to any one ofembodiments 1-20, or to any one of embodiments 36-37, which is stable at2-8° C. for 6-72 months.

Embodiment 31 is an immunogenic composition according to any one ofembodiments 1-20, or to any one of embodiments 36-37, which is stable at25° C. for at least six months.

Embodiment 32 is an immunogenic composition according to any one ofembodiments 1-20, or to any one of embodiments 36-37, which is stable at40° C. for at least one week, preferably at least two weeks.

Embodiment 33 is a method for storing an immunogenic compositioncomprising HIV gp140 protein, the method comprising providing aimmunogenic composition according to any one of embodiments 1-20, or toany one of embodiments 28-32, or to any one of embodiments 36-37 andstoring said composition at 2-8° C. for at least one day, e.g. at leastone week, e.g. at least one month, e.g. 1 day-72 months, e.g. 6-48months, e.g. 12-36 months, e.g. 18-30 months.

Embodiment 34 is a method for preparing a long-term, storage stableimmunogenic composition that comprises HIV gp140 protein, the methodcomprising: (i) providing an immunogenic composition that comprises0.05-5 mg/mL HIV gp140 protein, 2-15% (w/v) sorbitol, 0.01-0.05%polysorbate 20, 5-20 mM histine buffer pH 5.5-7.0, water, and preferably0.7-4.0 mg/mL aluminum phosphate), and (ii) storing said composition at2-8° C. for at least one week, e.g. at least one month, e.g. 1-72months, e.g. 6-48 months, e.g. 12-36 months, e.g. 18-30 months.

Embodiment 35 is use of an immunogenic composition according to any oneof embodiments 1-20, or to any one of embodiments 28-32, or to any oneof embodiments 36-37, for administering to a subject, preferably a humansubject, to induce an immune response against HIV, wherein theimmunogenic composition prior to said administering has been stored at2-8° C. for at least one day, e.g. at least one week, e.g. at least twoweeks, e.g. at least three weeks, e.g. at least one month, e.g. at leasttwo months, e.g. at least three months, e.g. at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.28. 29, 30, 31, 32, 33, 34, 35, 36 months.

Embodiment 36 is the immunogenic composition of any one of embodiments1-9, wherein the HIV gp140 protein comprises a mixture of an HIV gp140protein comprising the amino acid sequence of SEQ ID NO:1 and an HIVgp140 protein comprising the amino acid sequence of SEQ NO: 2.

Embodiment 37 is the immunogenic composition of embodiment 36, whereinthe HIV gp140 protein comprising the amino acid sequence of SEQ ID NO: 1and the HIV gp140 protein comprising the amino acid sequence of SEQ NO:2 are present in the mixture at a 1:1 ratio.

EXAMPLES Example 1: Formulation Development Study for HIV Clade C Gp140Protein

There are numerous possibilities for each component that can be includedin a protein formulation, e.g., buffer, sugar, pH value, surfactant etc.For example, different possibilities for buffers can include phosphate,acetate, HEPES, Tris, MOPS, etc.; different possibilities for aminoacids can include histidine, arginine, lysine, alanine, etc.; differentpossibilities for sugars can include sucrose, sorbitol, glycerol,mannitol, trehalose, etc.; and different possibilities for surfactantcan include polysorbate 20, polysorbate 80, Tween-20, Tween-80, etc.There are also many other types of excipients, and numerouspossibilities for each, that can further be included in a proteinformulation, such as osmolytes (e.g., glycine, proline, glycerol, urea,etc.), salts (e.g., sodium chloride, potassium chloride, sodium sulfate,etc.), carbohydrates (e.g., lactose), proteins and polymers (e.g., HSA,gelatin, PVP, PLGA, PEG, etc.), chelators and antioxidants (e.g., EDTA,DTPA, ethanol, etc.), preservatives (e.g., benzyl alcohol, m-cresol,phenol, etc.), etc. See, e.g., Kamerzell et al. Advanced Drug DeliveryReviews (2011) 63, 1118-1159. Accordingly, there are many differenttheoretical combinations of components that could be used to identify aformulation. However, the most suitable formulation is dependent uponthe particular protein in the formulation.

The inventors therefore set out to find improved formulations that couldmeet the complex requirements in the unpredictable art of proteinformulation, in particular, an improved HIV gp140 formulation thatenables drug product manufacturing meeting large late phase andcommercial scale demands, and includes aluminum phosphate adjuvant andgp140 protein to be stored as drug product in single vials atrefrigerated temperature, and prevents instability associated withcertain components present in currently used formulations. Theformulations were designed for storage in liquid form, but with thepotential to be stored in lyophilized form and reconstituted in liquidprior to injection.

Formulation screening studies were designed to evaluate differentbuffers (histidine, phosphate, and acetate), buffer strengths (10 mM to50 mM), sugars (sucrose and sorbitol from 2% to 12% w/v), surfactants(polysorbate 20 and polysorbate 80 from 0.02% to 1% w/v), and pH valuesfrom 4.5 to 7.5 on the stability of HIV clade C gp140 proteinformulations. The parameters were varied as shown in Table 1.

TABLE 1 HIV clade C gp140 protein formulation study design BufferHistidine Phosphate Acetate (His) (Pho) (Ace) pH 5.5-6.5 6.5-7.5 4.5-5.5Buffer 10 mM, 20 mM, or 50 mM Strength Sugar Sucrose or sorbitol at 2%or 12% (w/v) Surfactant Polysorbate 20 (PS20) or polysorbate 80 (PS80)at 0.02%, 0.05%, or 0.10% (w/v)

The formulations were prepared by adding the buffer components at the pHvalue to be tested, followed by addition of sugar and surfactant. The pHwas adjusted as necessary. Then, the clade C gp140 protein (SEQ IDNO: 1) was added to a concentration of 1 mg/mL or 0.2 mg/mL.

Formulation stability was analyzed using a SolvoVPE system to evaluateconcentration and turbidity. Samples of each formulation were analyzedat time 0 (T₀), and then after stressing at 40° C. for 24 hours (T₂₄).Formulation stability was also analyzed using dynamic light scattering(DLS).

SolvoVPE Analysis:

A SolvoVPE (C Technologies, Inc.; Bridgeport, N.J., USA) was used todetermine protein concentration by measuring the UV absorbance ofsamples of each formulation at 280 nm and 350 nm. The change inturbidity was determined from the difference in absorbance at 350 nmbetween T₀ and T₂₄ measurements. For desired formulations, any change inturbidity should preferably be as small as possible. Increases inturbidity indicate that the protein is precipitating out of solution,and that the formulation is thus less stable. The results of theSolvoVPE analysis are shown in FIG. 1.

The results of the SolvoVPE analysis show that the acetate bufferformulations had the highest increase in turbidity (average change of+0.339), whereas phosphate buffer formulations showed a moderateincrease (average change of +0.177), and histidine buffer formulationsshows almost no change in turbidity (average change of −0.010). Since alarge change in turbidity is undesirable, the turbidity data indicatethat histidine buffer is the most optimal buffer of those tested forimproving the stability of HIV gp140 protein.

Dynamic Light Scattering (DLS) Analysis:

DLS was used to evaluate the colloidal stability, and specificallywhether there was any protein aggregation in the protein formulations.The change in radius (R_(h)) from T₀ at 20° C. to T_(7 days) at 70° C.was measured. More specifically, the R_(h) was measured for the initialsample at 20° C. Then, the sample was heated to 70° C., and held at atemperature of 70° C. for 7 days before measuring the final R_(h). Thedifference in the initial R_(h) value and the final R_(h) values isplotted in FIG. 1B. A large change in R_(h) indicates that there isprotein aggregation, and that the formulation is thus less desirable.

The results of the DLS analysis show that formulations containingsorbitol had a lower change in R_(h) as compared to formulationscontaining sucrose. Since a larger change in R_(h) is undesirable, theDLS data indicate that sorbitol is the most optimal sugar of thosetested for improving the stability of HIV gp140 protein.

The results of the above described studies also indicated that thesugar, sugar concentration, surfactant, surfactant concentration andprotein concentration had an effect on the stability, so theseparameters were further investigated as described in Example 2 below.

Example 2: Effect of Sugar, Surfactant, and Protein Concentration onStability of HIV Clade C Gp140 Formulations

HIV gp140 protein immunogenic formulations were prepared in a 10 mMhistidine buffer, pH 6.0±0.5, with the following parameters beingvaried: sugar (sorbitol and sucrose), sugar concentration (2% and 12%w/v), polysorbate (PS20 and PS80), polysorbate concentration (0.02% and0.1% w/v), and protein concentration (0.2 mg/mL and 1.0 mg/mL). Theformulations were prepared by adding 10 mM histidine buffer, pH 6.0±0.5,followed by addition of sugar and surfactant. The pH was adjusted asnecessary. HIV gp140 clade C protein (SEQ ID NO: 1) was added to thedesired concentration. The formulations were then analyzed byHigh-Performance Size Exclusion Chromatography (HP-SEC), as describedbelow.

The formulations prepared and tested are shown below in Table 2.

TABLE 2 HIV gp140 protein formulations Sugar Surfactant Protein Concen-Concen- concen- tration tration tration Formulation Sugar (% w/v)Surfactant (% w/v) (mg/mL) 1 sorbitol 12 PS20 0.02 0.2 2 sorbitol 12PS80 0.1 0.2 3 sorbitol 2 PS20 0.1 0.2 4 sorbitol 2 PS20 0.1 0.2 5sorbitol 2 PS80 0.02 1 6 sorbitol 12 PS80 0.02 1 7 sorbitol 12 PS20 0.021 8 sorbitol 2 PS80 0.1 1 9 sorbitol 12 PS80 0.1 1 10 sucrose 12 PS800.02 0.2 11 sucrose 2 PS20 0.02 0.2 12 sucrose 12 PS80 0.1 0.2 13sucrose 2 PS80 0.02 0.2 14 sucrose 2 PS80 0.1 1 15 sucrose 2 PS20 0.02 116 sucrose 2 PS20 0.1 1 17 sucrose 12 PS20 0.1 1 18 sucrose 12 PS20 0.11

High-Performance Size Exclusion Chromatography (HP-SEC) Analysis:

HP-SEC was used to analyze the amount of hexamer, trimer, and high/lowmolecular weight species in formulation samples by monitoring theabsorbance at 280 nm. The HIV gp140 protein exists predominantly as atrimer. Some HIV gp140 protein hexamer species is also observed.Although the trimer species is the desired species, immunogenicityagainst HIV is observed for both the trimer species and the hexamerspecies. High molecular weight (HMW) and low molecular weight (LMW)species in the formulations are undesired. In particular, the presenceof LMW species indicates cleavage and/or degradation of the gp140protein. HMW species may be caused by a number of factors, such asaggregation of the gp140 protein. Samples of each formulation wereanalyzed at time 0 (T₀), and then stressed at 40° C. for 24 hours (T₂₄).Formulations containing 1 mg/mL clade C gp140 protein were dilutedtwo-fold before injection into the HP-SEC system, and formulationscontaining 0.2 mg/mL clade C gp140 protein were not diluted prior toinjection.

The results of the HP-SEC analysis are shown below in Table 3, and inFIGS. 2A-2F. The results indicate that amount of trimer and hexamer forboth T₀ and T₂₄ samples depends on the concentration of surfactant andHIV gp140 protein. Formulations having a lower surfactant concentration(0.02% w/v) had a higher observed value of trimer+hexamer species of theHIV gp140 protein, with less high/low molecular weight species observed(see FIGS. 2B, 2D, and 2F), both before and after sample stressing.Formulations having a higher HIV gp140 protein concentration (1.0 mg/mL)also had a higher observed value of trimer+hexamer species of the HIVgp140 protein, with less high/low molecular weight species observed (seeFIGS. 2B and 2D), both before and after sample stressing (see FIGS. 2Aand 2C). Formulations containing polysorbate 20, as opposed topolysorbate 80, also had a lower amount of lower molecular weightspecies present (see FIG. 2E).

TABLE 3 Results of HP-SEC Analysis. High Molecular Low Molecular WeightSpecies (%) Hexamer (%) Trimer (%) Weight Species (%) Formulation T₀ T₂₄T₀ T₂₄ T₀ T₂₄ T₀ T₂₄ 1 0.57 0 10.69 10.13 88.05 89.87 0 0 2 0.22 0 8.489.55 80.08 85.39 11.22 5.06 3 0.2 0 9.15 9.51 84.47 85.77 0 4.72 4 0 09.26 9.56 84.63 86.71 0 3.73 5 0 0 10.72 12.27 88.41 87.73 0.86 0 6 0 010.78 11.6 88.44 88.4 0.78 0 7 0 0 11.08 11.7 88.6 88.3 0.32 0 8 0 010.46 11.67 87.33 88.33 2.21 0 9 0 0 10.6 11.57 86.91 88.43 2.48 0 10 00 9.79 10.02 88.17 89.98 2.05 0 11 0 0 10.52 10.54 88.75 89.46 0 0 12 00 8.9 9.23 79.79 83.51 11.31 7.26 13 0 0 9.74 10.58 87.58 89.42 2.68 014 0 0 10.37 11.82 87.38 87.98 2.25 0.2 15 0 0 10.91 12.15 88.77 87.850.32 0 16 0 0 10.57 11.42 87.71 87.72 1.72 0.86 17 0 0 10.63 11.45 88.5188.43 0 0.12 18 0 0 10.74 11.54 88.28 88.46 0 0

The data shown in Table 3 indicates that the combination of sorbitol andhistidine buffer is preferable to the combination of histidine bufferand sucrose. For examples, the formulations containing histidine bufferand sorbitol, such as formulations 1, 4, and 7, showed the least changebetween T₀ and T₂₄ in the amount of hexamer and trimer species ascompared to that observed for the formulations containing histidinebuffer and sucrose. This result was surprising because histidine bufferand sorbitol are not typically used in combination for proteinformulations, whereas histidine buffer and sucrose are often used incombination. It was thus unexpected that the most optimal formulationcomprised histidine buffer (pH 6.0±0.5) and sorbitol, rather thanhistidine buffer and sucrose. The above study also indicates thatincluding polysorbate 20 as a surfactant (see, e.g., FIG. 2E) at aconcentration of 0.2% (w/v) (see, e.g., FIGS. 2B, 2D, and 2F) provides aformulation in which the stability of the HIV gp140 protein is furtherimproved.

Advantages of the formulations identified herein over the one that iscurrently used for gp140 drug product in clinical trials, include thatthey use existing qualified compendial grade excipients that are readilyavailable for large scale manufacturing and do not suffer from knownissues that might negatively influence stability in the long term, andthey enable storage of stable bulk drug substance and antigen drugproduct. Moreover, these formulations enable storage and stability ofadjuvanted drug product as a single vial drug product at refrigeratedtemperature. These formulations are suitable for storage in liquid form,but also have the potential to be stored in lyophilized form and thenreconstituted in liquid prior to injection, which is yet anotheradvantage.

Example 3: Stability Studies of HIV Mosaic Gp140 Protein Formulations

Two formulations containing HIV mosaic gp140 protein (SEQ ID NO: 2) wereexamined for stability in a freeze-thaw and temperature cycling study.The formulations tested are shown in Table 4

TABLE 4 HIV Mosaic gp140 Protein Formulations Formu- lation ProteinBuffer Sugar Surfactant F1 1.0 mg/mL 10 mM histidine, 12% sorbitol 0.02%PS20 pH 6.5 F2 1.0 mg/mL 10 mM histidine,  2% sorbitol 0.02% PS20 pH 6.5

The formulations were subjected to multiple freeze-thaw cycles. Onefreeze-thaw cycle was conducted by freezing at −80° C. or −40° C. for 24hours, followed by thawing at −2° C. to 8° C. for 24 hours. Samples wereanalyzed at the end of 1, 3, and 5 cycles of freeze-thaw by measuringthe absorbance at 280 nm (concentration) and 350 nm (turbidity). Theresults are shown in FIG. 3A and FIG. 3B.

The results show that the absorbance at 350 nm and 280 nm was largelyunchanged for both formulations F1 and F2 after multiple freeze-thawcycles, indicating that the concentration and turbidity of theformulation were relatively unaffected. This demonstrates that the HIVgp140 protein in the formulations is stable to freeze-thaw.

Example 4: Long Term Stability Study of HIV Gp140 Protein Formulations

The stability of HIV gp140 protein compositions in histidine buffer,both with and without adjuvant according to embodiments of the inventionwas compared to the stability of an HIV gp140 protein compositionformulated in HEPES buffer, both with and without adjuvant. Thecompositions tested are shown in Table 5. All formulations contained 0.2mg/mL HIV clade C gp140 protein (SEQ ID NO: 1).

TABLE 5 HIV gp140 protein formulations for long term stability study.Formulation Buffer Composition Adjuvant? 1 20 mM HEPES, pH 6.5 Noadjuvant 90 m, M NaCl 4% (w/v) sucrose 0.02% polysorbate 80 2 10 mMhistidine buffer, pH 6.5 No adjuvant 12% (w/v) sorbitol 0.02% (w/v)polysorbate 20 3 10 mM histidine buffer, pH 6.5 No adjuvant 2% (w/v)sorbitol 0.02% (w/v) polysorbate 20 4 10 mM histidine buffer, pH 6.5 Noadjuvant 5% (w/v) sorbitol 0.02% (w/v) polysorbate 20 5 20 mM HEPES, pH6.5 +aluminum phosphate 90 m, M NaCl adjuvant (0.85 mg/mL) 4% (w/v)sucrose 0.02% polysorbate 80 6 10 mM histidine buffer, pH 6.5 +aluminumphosphate 12% (w/v) sorbitol adjuvant (0.85 mg/mL) 0.02% (w/v)polysorbate 20 7 10 mM histidine buffer, pH 6.5 +aluminum phosphate 2%(w/v) sorbitol adjuvant (0.85 mg/mL) 0.02% (w/v) polysorbate 20 8 10 mMhistidine buffer, pH 6.5 +aluminum phosphate 5% (w/v) sorbitol adjuvant(0.85 mg/mL) 0.02% (w/v) polysorbate 20

Compositions were stored at 2° C. to 8° C.; 25° C. and 60% relativehumidity (RH); and 40° C. and 75% RH. The study is ongoing, and samplesare tested after storage for 2 weeks, 1, 2, 3, 6, 9, 12, 18, 24, 30, and36 months.

Samples were tested by reduced SDS after storage for up to three months.Under reducing conditions, as the SDS-PAGE was performed, the trimericand hexameric forms were reduced to a monomeric form that showed up asingle band on the gel. Any degradation of the protein to lowermolecular weight species resulted in a change (decrease) in themonomeric form of the protein observed on the gel. The data for samplesstored at 25° C. and 60% RH, and at 40° C. and 75% RH are shown in FIGS.4A-4D. The data show that all formulations were stable under storage at25° C. and 60% RH both with and without aluminum phosphate adjuvant forup to three months (FIGS. 4A and 4C). However, formulations containinghistidine buffer and sorbitol were more stable than the formulationcontaining HEPES and sucrose (FIGS. 4B and 4D) when stored at 40° C. and75% RH, both with and without aluminum phosphate adjuvant. In addition,after six months storage at 25° C. the histidine formulated materialwith 12% sorbitol, without aluminum phosphate adjuvant, showed betterstability compared to the HEPES buffered formulation as measured bySDS-PAGE (smaller change in % compared to initial measurement; data notshown). Moreover, gp140 that was formulated with histidine buffer,sorbitol and aluminum phosphate was observed to be stable at 25° C. forat least six months (data not shown). Also when formulated withhistidine buffer and sorbitol, at higher concentrations of aluminumphosphate adjuvant (3.84 mg/mL), the HIV gp140 protein was observed tobe stable at 25° C. for at least six months and at 40° C. for at leastthree months as measured by reduced SDS PAGE (less than 25% reduction,data not shown; while measurements using ELISA showed stability (lessthan 50% reduction) at 40° C. for at least two weeks, data not shown).These data further indicate that HIV gp140 protein formulationscontaining histidine buffer and sorbitol have enhanced stability, andcan thus be stored as an adjuvanted drug product in a single vial atelevated temperatures, which is an advantage as it is often difficult toformulate adjuvanted protein that is stable to storage at elevatedtemperatures. This means that the drug product with adjuvant can bestored in a single vial at a manufacturing or a fill-and-finish site,and does not require separate storage with mixing in a pharmacy or atbed-side just prior to administration. Apart from being economicallybeneficial, this is a huge advantage especially for an HIV vaccineproduct that is intended to be also used in many places where capacityand facilities may be limited. It is also an advantage of limiting thenumber of operations to be performed with the drug product on site wherea large number of subjects are to be vaccinated in a single campaign.Thus, the formulations of the invention surprisingly improve thepractical properties for use of the final vaccine, especially inresource-limited settings.

A combination of two HIV gp140 proteins, one having SEQ ID NO: 1 and onehaving SEQ ID NO: 2 is mixed (total protein concentration 0.2-1.0 mg/mL,e.g. 0.1 mg/mL for each protein resulting in a total concentration ofgp140 protein of 0.2 mg/mL) and tested in the preferred formulation,i.e. 2% to 15% (w/v) sorbitol; 0.01 to 0.05% (w/v), e.g. 0.02%,polysorbate 20; and 5 to 20 mM, e.g. 10 mM, histidine buffer at a pH of5.5 to 7.0, e.g. pH 6.5, and preferably aluminum phosphate, e.g. 0.7-4mg/mL, e.g. 0.85 or 3.84 mg/mL, using methods as described above. Thiscomposition is also expected to be stable for at least six months at2-8° C., like the individual proteins in this formulation as indicatedabove.

It is understood that the examples and embodiments described herein arefor illustrative purposes only, and that changes could be made to theembodiments described above without departing from the broad inventiveconcept thereof. It is understood, therefore, that this invention is notlimited to the particular embodiments disclosed, but it is intended tocover modifications within the spirit and scope of the invention asdefined by the appended claims.

REFERENCES

-   1. Abbink et al., Virol (2007) 81(9): 4654-63.-   2. Baicu et al., Cryobiology (2002) 45(1) 33-48.-   3. Barouch et al., Nat Med (2010), 16: 319-323.-   4. Barouch D H et al., Science (2015) 349: 320-324.-   5. Barouch et al., Cell (2013) 155: 1-9.-   6. Harris A et al., Proc Natl Acad Sci USA (2011) 108: 11440-11445.-   7. Hoganson et al., Bioprocessing (2002) J 1: 43-8.-   8. Kamerzell et al., Advanced Drug Delivery Reviews (2011) 63,    1118-1159.-   9. Kovacs J M et al., Proc Natl Acac Sci USA (2012) 109:    12111-12116.-   10. Lepe-Zuniga et al., J. Immunol. Methods (1987) 103(1), 145.-   11. Nkolola et al., J. Virol. (2014) 88(17), 9538-9552.-   12. Nkolola et al., J. Virol. (2010) 84(7), 3270-3279.-   13. Sanders R W et al., J. Virol. (2002) 76: 8875-8889.-   14. Sanders R W et al., Science (2015) 349(6244): aac4223, doi:    10.1126/science.aac4223.-   15. Uchiyama, Biochimica Biophysica Acta (2014) 1844, 2041-2052.-   16. Zhang C W-H et al., J Biol Chem (2001) 276: 39577-39585.-   17. Zigler et al., In Vitro Cell. Dev. Biol. (1985) 21(5), 282-287.-   18. WO 2010/042942-   19. WO 2014/107744-   20. US20120076812;-   21. WO 2016/049287-   22. WO 2010/059732-   23. US20120076812-   24. WO 2007/104792-   25. PCT/EP2016/081159

The invention claimed is:
 1. A composition comprising, relative to thetotal volume of the composition: a. an HIV gp140 protein or a mixture ofat least two HIV gp140 proteins; b. 2% to 15% (w/v) sorbitol; c. 0.01 to0.05% (w/v) polysorbate 20; and d. 5 to 20 mM histidine buffer at a pHof 5.5 to 7.0.
 2. The composition of claim 1, wherein a concentration ofthe HIV gp140 protein or of the mixture of HIV gp140 proteins is 0.05mg/mL to 5 mg/mL.
 3. The composition of claim 1, wherein a concentrationof the HIV gp140 protein or of the mixture of HIV gp140 proteins is 0.2mg/mL to 1 mg/mL.
 4. The composition of claim 1, wherein theconcentration of sorbitol is 5% (w/v).
 5. The composition of claim 1,wherein the concentration of sorbitol is 12% (w/v).
 6. The compositionof claim 1, wherein the concentration of polysorbate 20 is 0.02% (w/v).7. The composition of claim 1, wherein the concentration of thehistidine buffer is 10 mM, and the pH of the histidine buffer is 6.5. 8.The composition of claim 1, further comprising 0.7 mg/mL to 4.0 mg/mLaluminum phosphate adjuvant.
 9. The composition of claim 1, comprisingthe HIV gp140 protein having the amino acid sequence of SEQ ID NO: 1.10. The composition of claim 1, comprising the HIV gp140 protein havingthe amino acid sequence of SEQ ID NO:
 2. 11. The composition of claim 1,comprising the mixture of an HIV gp140 protein having the amino acidsequence of SEQ ID NO: 1 and an HIV gp140 protein having the amino acidsequence of SEQ NO:
 2. 12. The composition of claim 1, being a liquidcomposition.
 13. A composition comprising, relative to the total volumeof the composition, a. 0.2 mg/mL to 1 mg/mL of an HIV gp140 proteincomprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or ofa mixture of an HIV gp140 protein comprising the amino acid sequence ofSEQ ID NO:1 and an HIV gp140 protein comprising the amino acid sequenceof SEQ NO: 2; b. 5% or 12% (w/v) sorbitol; c. 0.02% (w/v) polysorbate20; d. 10 mM histidine buffer at a pH of 6.5; and e. 0.85 mg/mL to 3.84mg/mL of aluminum phosphate adjuvant.
 14. The composition of claim 13,being a liquid composition.
 15. The composition of claim 13, which isstable upon storage at 2-8° C. for at least six months.
 16. A method ofpreparing a composition comprising admixing: a. 0.2 mg/mL to 1 mg/mL ofan HIV gp140 protein or of a mixture of at least two HIV gp140 proteins;b. 2% to 15% (w/v) sorbitol; c. 0.01 to 0.05% (w/v) polysorbate 20; andd. 5 to 20 mM histidine buffer at a pH of 5.5 to 7.0; to thereby obtainthe composition.
 17. A method according to claim 16, further comprisingadmixing 0.7 mg/mL to 4.0 mg/mL aluminum phosphate adjuvant.
 18. Amethod for preparing a long-term, storage stable composition thatcomprises HIV gp140 protein, the method comprising: (i) providing acomposition according to claim 13, and (ii) storing said composition at2-8° C. for at least one week.
 19. A method of inducing an immuneresponse against HIV in a subject, the method comprising administeringto the subject a composition according to claim 1, wherein thecomposition prior to the administering has been stored at 2-8° C. forone day to 36 months.
 20. A method of inducing an immune responseagainst HIV in a subject, the method comprising administering to thesubject a composition according to claim 12, wherein the compositionprior to the administering has been stored at 2-8° C. for one day to 36months.
 21. The method of claim 19, wherein the composition isadministered to the subject in combination with a second compositioncomprising adenovirus 26 vectors encoding one or more additional HIVantigens having the amino acid sequences of one or more of SEQ ID NOs:3-12.